DESCRIPTION: This revised application describes studies to elucidate the functional role and molecular mechanism of action of tif, a new member of the axl family of receptors. Tif, which was cloned by RTPCR from a K562 library, is an extremely interesting receptor in that it bears extracellular fibronectin domains as well as Ig domains, suggesting a role in cell-matrix attachment and signalling. In addition, its pattern of RNA expression, gonad>brain>other tissues, suggests a potential role in primitive cell maintenance and self-renewal. In this application, the Applicant proposes to: 1) Study the transforming potential of tif by enforced over- expression; 2) Investigate the role of tif in hematopoietic determination and differentiation by analyzing the phenotype of both K562 and Ba/F3 cells constitutively expressing activated tif, and by differentiating ES cells with a targeted gene disruption of the tif locus; 3)Identify the immediate downstream components of the tif signaling pathway via immonocoprecipitation and via the yeast two-hybrid system; 4)Search for a functional interaction between tif and the Epo receptor by enforced expression of an EpoR/tif chimera, analyzing signal transduction and cellular phenotype in transfected Ba/F3 and K562 cells; and 5)Identify and clone the tif ligand via expression or affinity column chromatography. These studies are designed to provide the first important steps towards understanding the role of Tif in hematopoietic differentiation.